Single-Molecule Super-Resolution Light-Sheet Microscopy

ChemPhysChem ◽  
2014 ◽  
Vol 15 (4) ◽  
pp. 577-586 ◽  
Author(s):  
Ying S. Hu ◽  
Maxwell Zimmerley ◽  
Yu Li ◽  
Robin Watters ◽  
Hu Cang
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Light Sheet ◽  
Light Sheet Microscopy

scholarly journals Recent Progress in Light Sheet Microscopy for Biological Applications

2018 ◽  
Vol 72 (8) ◽  
pp. 1137-1169 ◽  
Author(s):  
Krishnendu Chatterjee ◽  
Feby Wijaya Pratiwi ◽  
Frances Camille M. Wu ◽  
Peilin Chen ◽  
Bi-Chang Chen
Keyword(s):  
Developmental Biology ◽  
Fluorescence Microscopy ◽  
Single Cell ◽  
Single Molecule ◽  
Super Resolution ◽  
Light Sheet ◽  
High Signal ◽  
Spatiotemporal Resolution ◽  
Light Sheet Microscopy ◽  
Resolution Imaging

The introduction of light sheet fluorescence microscopy (LSFM) has overcome the challenges in conventional optical microscopy. Among the recent breakthroughs in fluorescence microscopy, LSFM had been proven to provide a high three-dimensional spatial resolution, high signal-to-noise ratio, fast imaging acquisition rate, and minuscule levels of phototoxic and photodamage effects. The aforementioned auspicious properties are crucial in the biomedical and clinical research fields, covering a broad range of applications: from the super-resolution imaging of intracellular dynamics in a single cell to the high spatiotemporal resolution imaging of developmental dynamics in an entirely large organism. In this review, we provided a systematic outline of the historical development of LSFM, detailed discussion on the variants and improvements of LSFM, and delineation on the most recent technological advancements of LSFM and its potential applications in single molecule/particle detection, single-molecule super-resolution imaging, imaging intracellular dynamics of a single cell, multicellular imaging: cell–cell and cell–matrix interactions, plant developmental biology, and brain imaging and developmental biology.

scholarly journals 3D single-molecule super-resolution microscopy with a tilted light sheet

2017 ◽  
Author(s):  
Anna-Karin Gustavsson ◽  
Petar N. Petrov ◽  
Maurice Y. Lee ◽  
Yoav Shechtman ◽  
W. E. Moerner
Keyword(s):  
Single Molecule ◽  
Mammalian Cells ◽  
Double Helix ◽  
Super Resolution ◽  
Nuclear Lamina ◽  
Light Sheet ◽  
Point Spread Functions ◽  
Light Sheet Microscopy ◽  
Point Spread ◽  
Resolution Imaging

Tilted light sheet microscopy with 3D point spread functions (TILT3D) combines a novel, tilted light sheet illumination strategy with long axial range point spread functions (PSFs) for low-background, 3D super-localization of single molecules as well as 3D super-resolution imaging in thick cells. Because the axial positions of the single emitters are encoded in the shape of each single-molecule image rather than in the position or thickness of the light sheet, the light sheet need not be extremely thin. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The result is simple and flexible 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validated TILT3D for 3D super-resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed Tetrapod PSFs for fiducial bead tracking and live axial drift correction.

scholarly journals Single-Molecule Light-Sheet Microscopy with Local Nanopipette Delivery

2021 ◽  
Vol 93 (8) ◽  
pp. 4092-4099
Author(s):  
Bing Li ◽  
Aleks Ponjavic ◽  
Wei-Hsin Chen ◽  
Lee Hopkins ◽  
Craig Hughes ◽  
...  
Keyword(s):  
Single Molecule ◽  
Light Sheet ◽  
Light Sheet Microscopy

scholarly journals Circumvention of common labeling artifacts using secondary nanobodies

2019 ◽  
Author(s):  
Shama Sograte-Idrissi ◽  
Thomas Schlichthaerle ◽  
Carlos J. Duque-Afonso ◽  
Mihai Alevra ◽  
Sebastian Strauss ◽  
...  
Keyword(s):  
Super Resolution ◽  
Light Sheet ◽  
Common Procedure ◽  
Tissue Samples ◽  
Localization Accuracy ◽  
Light Sheet Microscopy ◽  
Large Size ◽  
Resolution Imaging ◽  
Single Domain Antibodies ◽  
Secondary Antibodies

AbstractThe most common procedure to reveal the location of specific (sub)cellular elements in biological samples is via immunostaining followed by optical imaging. This is typically performed with target-specific primary antibodies (1.Abs), which are revealed by fluorophore-conjugated secondary antibodies (2.Abs). However, at high resolution this methodology can induce a series of artifacts due to the large size of antibodies, their bivalency, and their polyclonality. Here we use STED and DNA-PAINT super-resolution microscopy or light sheet microscopy on cleared tissue to show how monovalent secondary reagents based on camelid single-domain antibodies (nanobodies; 2.Nbs) attenuate these artifacts. We demonstrate that monovalent 2.Nbs have four additional advantages: 1) they increase localization accuracy with respect to 2.Abs; 2) they allow direct pre-mixing with 1.Abs before staining, reducing experimental time, and enabling the use of multiple 1.Abs from the same species; 3) they penetrate thick tissues efficiently; and 4) they avoid the artificial clustering seen with 2.Abs both in live and in poorly fixed samples. Altogether, this suggests that 2.Nbs are a valuable alternative to 2.Abs, especially when super-resolution imaging or staining of thick tissue samples are involved.

scholarly journals Single Molecule Imaging in Live Embryos Using Lattice Light-Sheet Microscopy

2018 ◽  
pp. 541-559 ◽  
Author(s):  
Mustafa Mir ◽  
Armando Reimer ◽  
Michael Stadler ◽  
Astou Tangara ◽  
Anders S. Hansen ◽  
...  
Keyword(s):  
Single Molecule ◽  
Light Sheet ◽  
Single Molecule Imaging ◽  
Light Sheet Microscopy

scholarly journals Virtual-'Light-Sheet' Single-Molecule Localisation Microscopy Enables Quantitative Optical Sectioning for Super-Resolution Imaging

PLoS ONE ◽  
2015 ◽  
Vol 10 (4) ◽  
pp. e0125438 ◽  
Author(s):  
Matthieu Palayret ◽  
Helen Armes ◽  
Srinjan Basu ◽  
Adam T. Watson ◽  
Alex Herbert ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Light Sheet ◽  
Optical Sectioning ◽  
Resolution Imaging ◽  
Localisation Microscopy

scholarly journals 3D Interferometric Lattice Light-Sheet Imaging

2020 ◽  
Author(s):  
Bin Cao ◽  
Guanshi Wang ◽  
Jieru Li ◽  
Alexandros Pertsinidis
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Light Sheet ◽  
Detection Sensitivity ◽  
Live Cells ◽  
Background Suppression ◽  
Axial Resolution ◽  
Volumetric Imaging ◽  
Spatio Temporal ◽  
And Function

Understanding cellular structure and function requires live-cell imaging with high spatio-temporal resolution and high detection sensitivity. Direct visualization of molecular processes using single-molecule/super-resolution techniques has thus been transformative. However, extracting the highest-resolution 4D information possible from weak and dynamic fluorescence signals in live cells remains challenging. For example, some of the highest spatial resolution methods, e.g. interferometric (4Pi) approaches1-6 can be slow, require high peak excitation intensities that accelerate photobleaching or suffer from increased out-of-focus background. Selective-plane illumination (SPIM)7-12 reduces background, but most implementations typically feature modest spatial, especially axial, resolution. Here we develop 3D interferometric lattice light-sheet (3D-iLLS) imaging, a technique that overcomes many of these limitations. 3D-iLLS provides, by virtue of SPIM, low light levels and photobleaching, while providing increased background suppression and significantly improved volumetric imaging/sectioning capabilities through 4Pi interferometry. We demonstrate 3D-iLLS with axial resolution and single-particle localization precision down to <100nm (FWHM) and <10nm (1σ) respectively. 3D-iLLS paves the way for a fuller elucidation of sub-cellular phenomena by enhanced 4D resolution and SNR live imaging.

scholarly journals Single-molecule light-sheet microscopy with local nanopipette delivery

2020 ◽  
Author(s):  
B. Li ◽  
A. Ponjavic ◽  
W. H. Chen ◽  
L. Hopkins ◽  
C. Hughes ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Single Molecule ◽  
Single Molecules ◽  
Light Sheet ◽  
Local Delivery ◽  
Live Cells ◽  
Biological Processes ◽  
Light Sheet Microscopy ◽  
Wide Range

AbstractDetection of single molecules in biological systems has rapidly increased in resolution over the past decade. However, delivery of single molecules has remained a challenge. Currently there is no effective method that can both introduce a precise amount of molecules onto or into a single cell at a defined position, and then image the cellular response. Here we have combined light sheet microscopy with local delivery, using a nanopipette, to accurately deliver individual proteins to a defined position. We call this method local delivery selective plane illumination microscopy (ldSPIM). ldSPIM uses a nanopipette and the ionic feedback current at the nanopipette tip to control the position from which molecules are delivered. The number of proteins delivered can be controlled by varying the voltage applied. For single-molecule detection, we implemented single-objective SPIM using a reflective atomic force microscopy cantilever to create a 2µm thin sheet. Using this setup, we demonstrate that ldSPIM can deliver single fluorescently-labeled proteins onto the plasma membrane of HK293 cells or into the cytoplasm. Next, we deposited aggregates of amyloid-β, which causes proteotoxicity relevant to Alzheimer’s disease, onto a single macrophage stably expressing a MyDD88-eGFP fusion construct. Whole-cell imaging in 3D mode enables live detection of MyDD88 accumulation and formation of MyDDosome signaling complexes, as a result of aggregate-induced triggering of toll-like receptor 4. Overall, we demonstrate a novel multifunctional imaging system capable of precise delivery of single proteins to a specific location on the cell surface or inside the cytoplasm and high-speed 3D detection at single-molecule resolution within live cells.Statement of SignificanceThis paper describes and validates a new method to study biological processes based on the controlled local delivery of molecules onto or into the cell, combined with single molecule imaging using light sheet microscopy. we not only demonstrate the instrument’s capability of delivering controlled numbers of molecules to a defined position, down to the level of single molecules, but also its potential in study of the triggering of the innate immune response by protein aggregates, a key process in the development of neurodegenerative diseases such as Alzheimer’s disease. The same approach could be applied to a wide range of other important biological processes allowing them to be followed in live cells in real-time, hence it will be of great interest to the biophysical community.

scholarly journals A polymer gel index-matched to water enables diverse applications in fluorescence microscopy

2020 ◽  
Author(s):  
Xiaofei Han ◽  
Yijun Su ◽  
Hamilton White ◽  
Kate M. O’Neill ◽  
Nicole Y. Morgan ◽  
...  
Keyword(s):  
Surface Passivation ◽  
Super Resolution ◽  
Light Sheet ◽  
Polymer Gel ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Neural Structure ◽  
Uv Curable ◽  
Isotropic Resolution ◽  
Light Sheet Microscopy

AbstractWe demonstrate diffraction-limited and super-resolution imaging through thick layers (tens-hundreds of microns) of BIO-133, a biocompatible, UV-curable, commercially available polymer with a refractive index (RI) matched to water. We show that cells can be directly grown on BIO-133 substrates without the need for surface passivation and use this capability to perform extended time-lapse volumetric imaging of cellular dynamics 1) at isotropic resolution using dual-view light-sheet microscopy, and 2) at super-resolution using instant structured illumination microscopy. BIO-133 also enables immobilization of 1) Drosophila tissue, allowing us to track membrane puncta in pioneer neurons, and 2) Caenorhabditis elegans, which allows us to image and inspect fine neural structure and to track pan-neuronal calcium activity over hundreds of volumes. Finally, BIO-133 is compatible with other microfluidic materials, enabling optical and chemical perturbation of immobilized samples, as we demonstrate by performing drug and optogenetic stimulation on cells and C. elegans.

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